Recent studies from our laboratory uncovered a novel mechanism for cell invasion by the protozoan Trypanosoma cruzi. Prior to entry into non- phagocytic mammalian cells, host lysosomes are recruited to the parasite attachment site and gradually fuse with the plasma membrane, originating the parasite-containing vacuole. In our search for signal transduction events involved in this process, we found that infective trypomastigotes mobilize Ca2 plus from host cell intracellular stores, inducing transient increases in the cytosolic free Ca2 plus concentration of a variety of mammalian cell types. Rapid rearrangements in the host cell cortical actin cytoskeleton, a condition known to result in enhanced T. cruzi invasion, are observed as a consequence of trypomastigote-induced Ca2 plus-transients. Prevention of these transients significantly inhibits host cell invasion by the parasites. We now plan to directly investigate the link between the T. cruzi-induced intracellular free Ca2 plus transients and the mobilization and fusion of lysosomes with the plasma membrane in mammalian cells. Specifically, we will: 1) using a video-microscopy assay, examine the role of host cell cytosolic free Ca2 plus transients on the recruitment and/or plasma membrane fusion of lysosomes induced by T. cruzi; 2) investigate the role of the major PKC substrate, Ca2 plus-calmodulin and actin-binding protein MARCKS in the host cell events leading to lysosome fusion and T. cruzi invasion; 3) use a permeabilized mammalian cell system to investigate the molecular mechanisms involved in Ca2 plus-regulated lysosome exocytosis, specifically the role of the NSF/SNAP/SNARE fusion complex, synaptotagmin, annexins and monomeric and trimeric GTP-binding proteins; 4) correlate the data obtained in the lysosome exocytosis assay with T. cruzi invasion, using specific inhibitors and cellular microinjection. These studies will advance our knowledge of the unusual cell invasion mechanism utilized by the important human pathogen T. cruzi, as well as of fundamental membrane traffic events involving lysosomes in mammalian cells.